中华绒螯蟹羧酸酯酶基因克隆及其在农药胁迫下的表达变化

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摘要：【目的】明確中华绒螯蟹羧酸酯酶基因（ES-CXEs）在中华绒螯蟹不同组织及在农药胁迫下的应激表达模式，为揭示其代谢解毒机制打下理论基础。【方法】采用RACE克隆ES-CXEs基因全长序列，并进行生物信息学分析;以实时荧光定量PCR检测中华绒螯蟹不同组织及在农药胁迫下ES-CXEs基因的表达变化。【结果】从中华绒螯蟹肝胰腺中克隆获得两段ES-CXEs基因cDNA序列（ES-CXE5和ES-CXE6），其中，ES-CXE5基因序列全长1889 bp（GenBank登录号MH201558），包括132 bp的5"端非编码区（5"UTR）、122 bp的3"端非编码区（3"UTR）和1635 bp的开放阅读框（ORF），推测其编码544个氨基酸序列;ES-CXE6基因序列全长3089 bp（GenBank登录号MH201555），包括282 bp的5"UTR、779 bp的3"UTR和2028 bp的ORF，推测其编码675个氨基酸序列。多序列比对分析结果显示，ES-CXE5和ES-CXE6基因均具有CXEs家族的特征序列，即催化三联体结构（S-E-H）和N-糖基化位点。ES-CXE5氨基酸序列与三疣梭子蟹、日本长额虾等甲壳类CXEs聚为一簇，而ES-CXE6氨基酸序列与水蚤类和芜菁叶蜂的CXEs聚为一簇。组织特异性表达分布显示，ES-CXEs基因在雌、雄性中华绒螯蟹不同组织中的相对表达量无显著差异（P>0.05），且在肝胰腺、肌肉、精巢和副性腺中均有表达，以肝胰腺中的相对表达量最高。在安全浓度的阿维菌素、敌百虫和高效氯氰菊酯胁迫下，ES-CXEs基因在中华绒螯蟹肝胰腺中的相对表达量呈明显诱导表达趋势，且ES-CXE5基因的相对表达量高于ES-CXE6基因。【结论】从中华绒螯蟹肝胰腺中克隆获得的ES-CXE5和ES-CXE6基因属于CXEs基因家族，在肝胰腺中高表达，且在农药胁迫下这两个基因的表达呈明显诱导表达趋势而参与杀虫剂的代谢解毒过程。

关键词： 中华绒螯蟹;羧酸酯酶（CXEs）;克隆;杀虫剂;代谢解毒

中图分类号： S966.16                            文献标志码： A 文章编号：2095-1191（2019）05-1093-11

Abstract：【Objective】Expression patterns of Eriocheir sinensis carboxylesterase gene（ES-CXEs）in various tissues and under pesticide tress were studied to provide theoretical basis for revealing its metabolic detoxification mechanism. 【Method】The full sequence of the ES-CXEs gene was cloned by using rapid-amplification of cDNA ends（RACE） technolo-gy and bioinformatics analysis was also conducted. Real-time fluorescence quantitative PCR was used for its expression analysis in various tissues and after pesticide treatment. 【Result】Two cDNA full-length sequences of ES-CXEs genes were cloned from E. sinensis hepatopancreas（named ES-CXE5 and ES-CXE6）. Full-length sequence of ES-CXE5 was 1889 bp（GenBank accession number：MH201558）， consisting of a 5"-untranslated region（UTR） of 132 bp， a 3"-UTR of 122 bp and an open reading frame（ORF） of 1635 bp. The deduced protein had 544 amino acids sequences. Full-length sequence of ES-CXE6 was 3089 bp（GenBank accession number：MH201555）， consisting of a 5"-UTR of 282 bp， a 3"-UTR of 779 bp and an ORF of 2028 bp. The deduced protein had 675 amino acids sequences. Multiple alignments revealed that both ES-CXE5 andES-CXE6 contained typical sequence of the CXEs family， which was catalytic triad structure and N-glycosylation site. Amino acid sequence of ES-CXE5 were clustered with Portunus trituberculatus，Pandalopsis japonica and other crustaceans. Amino acid sequence of ES-CXE6 were clustered with Daphnia magna and Athalia rosae. Tissue speci-fic-expression analysis showed that the relative expression level of two ES-CXEs genes were not significantly different in different tissue of female and male E. sinensis（P>0.05） and they were expressed in gill， hepatopancreas， muscle， testis and accessory gonads， with the highest relative expression in hepatopancreas. The expression levels of the two ES-CXEs genes in the hepatopancreas of E. sinensis exposed to low doses of avermectin，trichlorfon and β-cypermethrin were greatly induced， and the relative expression of ES-CXE5 were higher than that of ES-CXE6. 【Conclusion】ES-CXE5 and ES-CXE6 genes， cloning from the hepatopancreas of E. sinensis，belong to CXEs family，highly express in hepatopancreas， and their expression levels under pesticide stress are induced，thus they involve in the detoxification of the pesticides.

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